The principal goal of this NCDDG application is to investigate a novel antiviral strategy that involves the destruction of retroviral particles "from within." This strategy, first reported by our collaborators Drs. Natsoulis and Boeke, entails the targeting of deleterious enzymes (e.g. nucleases) to retrovirus particles via attachment to the viral coat protein. The capsid portion facilitates intracellular transport and assembly into virions, while the nuclease destroys infectivity by degrading the viral RNA. As part of this NCDDG application, we propose to investigate the utility of the HIV/SIV accessory proteins vpx and vpr for the same purpose. These two proteins represent excellent candidates for virion targeted drug delivery, since they are small, present in virus particles in quantities comparable to gag proteins, and dispensable for viral replication in vitro. Moreover, we have already shown that at least one of them (vpx) can be used to target "foreign" proteins to the virus particle in the context of an N-terminal fusion protein (see Preliminary Results Section). To investigate the utility of vpx and vpr as mediators of antiviral therapy, we propose in this project to define their as yet unknown mechanism(s) of action, to study their biosynthesis, and to elucidate their packaging requirements. In addition, we will explore genetic manipulation of vpx and vpr for the construction of fusion proteins with virion packaging ability. We will employ the SIVsm PBj virus system so that functions of native and genetically altered vpx and vpr proteins can be assessed in vitro and in vivo. Specific aims of the project are (i) define the precise stage in the viral replication cycle wherein vpx and vpr (alone and in combination) exert their biological activity, and determine what that activity is; (ii) evaluate whether vpx and vpr facilitate reverse transcription within viral particles; (iii) determine the composition and biosynthesis of a novel virion associated 30 kDA protein that has vpx specific immunoreactivity; and (iv) determine whether vpx and vpr can be utilized to target agents with potential antiviral activity to HIV and SIV virions. To accomplish these aims, we have begun to collaborate with Drs. Boeke and Natsoulis who have extensive experience in the area of capsid targeted viral inactivation using staphylococcal nuclease and barnase. This multidisciplinary effort will yield new insights into the mechanism of action of vpx and vpr, and will determine whether accessory proteins are suitable mediators of anti- HIV therapy.